Immunoglobulin peptides against heated bovine blood

ABSTRACT

The present invention is related to immunoglobulin peptides that recognize a thermostable antigen from bovine blood. The invention also provides methods for determining the presence of bovine blood in a food sample or an animal feed sample.

REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. patentapplication Ser. No. 11/616,427, filed Dec. 27, 2006, now U.S. Pat. No.7,696,329, which claims priority to Provisional Patent Application Ser.No. 60/805,685, filed on Jun. 23, 2006, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is directed to immunoglobulin peptides whichrecognize a thermostable antigen from bovine blood. The invention alsorelates to a kit containing one or more of such peptides. Furtherprovided are methods for determining the presence of bovine blood in afood sample or an animal feed sample.

The present invention is directed to immunoglobulin peptides whichrecognize a thermostable antigen from bovine blood. The invention alsorelates to a kit containing one or more of such peptides. Furtherprovided are methods for determining the presence of bovine blood in afood sample or an animal feed sample.

BACKGROUND OF THE INVENTION

Transmissible spongiform encephalopathy (TSE) agents induce fatalneurodegenerative diseases in mammalian species and humans. The TSEgroup in animals includes scrapie in sheep and goat, bovine spongiformencephalopathy (BSE) in cattle, feline spongiform encephalopathy,transmissible mink encephalopathy, and chronic wasting disease (CWD) inwild ruminants. Among them, BSE, commonly known as mad cow disease, hasbrought enormous economic consequences since its first incidence in theUnited Kingdom in 1986. The BSE outbreaks peaked in the United Kingdomin 1993 at almost 1000 new cases per week, and it caused more than182,000 cases between 1988 and 2002. In addition, the emergence of a newvariant form of Creutzfeldt-Jakob Disease (vCJD) in humans in the UnitedKingdom has been proposed to be possibly linked with BSE.

Meat and bone meal, an ingredient of animal feed, contaminated with aTSE agent was believed to be the major vehicle of BSE transmission,according to epidemiological inquiry. Meat and bone meal has beenproduced by rendering the discarded animal fat, bones, offal, and wholecarcasses from bovine, ovine, porcine, and poultry. Although the use ofmeat and bone meal in cattle as a nitrogen supplement had been a commonpractice for several decades, changes in rendering operations in the1970s and 1980s may have allowed the survival of the contagious agentsthat can be transmitted to the cattle through the meat and bone meal.The oral route was the major mode of natural transmission of BSE tocattle. To prevent the spread of BSE, the European Union in 1988 bannedthe inclusion of ruminant-derived proteins in animal feed. The U.S. Foodand Drug Administration also introduced the feed ban in 1997 to prohibitthe use of proteins derived from mammalian tissues in feeding ruminants.Nevertheless, the use of mammalian blood and blood products and anyproducts having only protein of porcine or equine origin in ruminantfeed products is still allowed. However, recent evidence indicates thatblood also carries some level of infectivity for TSE since transmissionof TSE has been demonstrated through inoculation of blood in animalsinfected with various strains of TSE (see, e.g., Castilla et al., Nat.Med., Vol. 11, pp. 982-985, 2005; Houston et al., Lancet, Vol. 356, pp.999-1000, 2000; and Taylor et al., J. Hosp. Infect., Vol. 46, pp. 78-79,2000). In addition to being used in animal feed, animal blood is used asa source of human food in many countries, usually in the form of bloodsausages, pudding, soup, bread and crackers. Even in the U.S., blood isused, e.g., in sausage products to enhance color, in bakery products asan egg substitute or in liquid foods as a clarifying agent.

Although prohibited, meat and bone meal from ruminant origin may stillenter cattle diets accidentally as a result of cross-contaminationduring feed mixing at the feed mills, transportation, storage, or thefarm. Indeed, the U.S. Food and Drug Administration found very lowlevels of prohibited meat and bone meal residues in the feedlotresulting from misformulation of the animal feed supplement at feedmills. In addition, although bovine blood and plasma are currentlyacceptable for certain uses, their future is uncertain due to changingattitudes of producers, blenders, and consumers who would like to haveproducts that are “free” of bovine blood and plasma products. Feedblenders and manufacturers usually acquire these blood and plasmaproducts from renderers who process or transport products from bothruminants (cattle, buffalo, sheep, goats, deer, elk, and antelopes) andnonruminants (pigs, and horses). Thus, care must be taken to ensure thatcross-contamination does not occur in the blending or manufacturingprocess.

Tools that permit enforcement of the meat and bone meal bans toeradicate BSE are becoming increasingly important for compliance withanimal byproduct regulation. Furthermore, the accurate labeling of meatproducts is mandated and monitored by the United States Department ofAgriculture (USDA) as well as by state and local governments. Mixingundeclared species in meat products is illegal under Food LabelingRegulations.

To date, there are technical limitations in detecting prohibitedresidues in animal feed because most of the analytical methods cannotdistinguish between allowable and prohibited bovine materials. Severalmethods have been developed to identify meat species includingelectrophoresis, chromatography, DNA hybridization, and immunoassays.Immunological techniques, including agar-gel immunodiffusion (AGID) andenzyme-linked immunosorbent assay (ELISA) are most commonly applied formeat species identification.

There are several disadvantages to the AGID method. Concentratedantiserum preparations are required to obtain visible precipitin linesin AGID. Obtaining the antiserum is expensive in large-scale testing.Furthermore, the sensitivity of AGID is variable. Usually ten percent ormore contamination must be present to detect adulteration with thismethod. Lastly, AGID cannot be used for species identification in cookedmeat because of the shortage of commercial antiserum specific to cookedmeats.

With respect to blood protein detection, the currently used methods suchas spectrophotometry, the Takayama confirmatory test and immunochemicalmethods also exhibit several limitations. This is in part due to thefact that these assays are mainly based on heat-labile blood proteins,resulting in lack of species specificity and making them unusable fordetection of blood in heat-treated samples such as cooked food or bloodmeal in animal feed.

BRIEF SUMMARY OF THE INVENTION

Among the various aspects of the present invention are immunoglobulinpeptides which bind an antigen from bovine blood. The immunoglobulinpeptides may be used, for example, in a screening assay to identify ordetect exogenous blood.

Another aspect of the present invention is a method for determining thepresence of bovine blood in a sample. The method comprises combining thesample with an immunoglobulin peptide which binds an antigen from bovineblood and determining whether any antigen from the sample was bound bythe immunoglobulin peptide.

Still another aspect of the present invention is a method fordetermining presence of bovine blood in a food sample or an animal feedsample, which comprises coating a carrier surface with a samplepotentially containing bovine blood antigen; contacting the coatedsurface with an immunoglobulin peptide having affinity for athermostable bovine blood antigen; and determining whether theimmunoglobulin peptide bound to the coated surface.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the effect of antigen coating (0.5 μg/100μL) on signals for commercial feeds using 1B4 antibody as more fullydescribed in Examples 2 and 3. Dilutions of 1B4 and secondary antibodyanti-IgG-HRP(Fc) were performed at 1:3 in 1% BSA-PBST.

FIG. 2 is a graph depicting species specificity of antibodies 3D6 and6G12 when used in a sandwich ELISA as more fully described in Examples 2and 3. Bba=autoclaved bovine blood, Sba=autoclaved sheep blood,Rba=autoclaved rabbit blood, Hba=autoclaved horse blood, Cba=autoclavedchicken blood, Pba=autoclaved pork blood, Tba=autoclaved turkey blood,NSB1=non-specific binding 1 (antibody only), NSB2=non-specific binding 2(antigen only), Blk=blank

FIGS. 3A and 3B are graphs depicting the effect of heat treatment on thespecies specificity of antibodies 6G12 (capture antibody) and 3D6(biotin conjugated detection antibody) when used in a sandwich ELISAwith autoclaved, cooked and raw meat as more fully described in Examples2 and 3. Bb=bovine blood, Sba=sheep blood, Rba=rabbit blood, Hba=horseblood, Cba=autoclaved chicken blood, Pba=pork blood, Tba=turkey blood,NSB1=non-specific binding 1 (antibody only), NSB2=non-specific binding 2(antigen only), Blk=blank

FIG. 4 is a graph depicting the cross-reactivity antibodies 6G12(capture antibody) and 3D6 (biotin conjugated detection antibody) whenused in a sandwich ELISA with cooked and autoclaved meat (flesh)proteins as more fully described in Examples 2 and 3. B=beef, S=sheep,R=rabbit, C=chicken, P=pork and T=turkey.

FIGS. 5A and 5B are graphs depicting the cross reactivity of antibodies6G12 (capture antibody) and 3D6 (biotin conjugated detection antibody)when used in a sandwich ELISA with non-flesh proteins as more fullydescribed in Examples 2 and 3. G=gelatin, S or Sp=soy proteinconcentrate, Ea=egg albumin, NFDM-non-fat dry milk, BSA=bovine serumalbumin, NSB1=non-specific binding 1 (antibody only), NSB2=non-specificbinding 2 (antigen only), Blk=blank

FIG. 6 is a graph depicting the cross reactivity of antibodies 6G12(capture antibody) and 3D6 (biotin conjugated detection antibody) whenused in a sandwich ELISA with flesh proteins as more fully described inExamples 2 and 3. Bm=bovine meat, Sm=sheep meat, Rm=rabbit meat,Hm=horse meat, Cm=chicken meat, Pm=porcine meat, Tm=turkey meat,NSB1=non-specific binding 1 (antibody only), NSB2=non-specific binding 2(antigen only), Blk=blank

FIGS. 7A and 7B are graphs depicting the performance of antibodies 6G12(capture antibody) and 3D6 (biotin conjugated detection antibody) whenused in a sandwich ELISA with commercial feed stuffs as more fullydescribed in Examples 2 and 3. Bbp=whole bovine blood powder,Bpm=spray-dried bovine plasma, Ppm=spray-dried porcine plasma,Smbm=sheep meat bone meal, Bmbm=bovine meat bone meal, Pmbm porcine meatbone meal, Fm=feather meal.

FIGS. 8A and 8B are graphs depicting the detection of autoclaved bovineblood in autoclaved porcine blood by antibodies 6G12 (capture antibody)and 3D6 (biotin conjugated detection antibody) when used in a sandwichELISA as more fully described in Examples 2 and 3.

FIG. 9 is a graph depicting the detection of bovine plasma meal inautoclaved bovine blood by antibodies 6G12 (capture antibody) and 3D6(biotin conjugated detection antibody) when used in a sandwich ELISA asmore fully described in Examples 2 and 3.

FIG. 10 is a graph depicting the detection of bovine plasma meal inautoclaved porcine blood by antibodies 6G12 (capture antibody) and 3D6(biotin conjugated detection antibody) when used in a sandwich ELISA asmore fully described in Examples 2 and 3.

FIG. 11 is a graph depicting the effect of heat treatment on the speciesspecificity of antibody 6E1 when used in an ELISA with soluble proteinsamples from autoclaved, cooked and raw meat as more fully described inExamples 2 and 3. Bb=bovine blood, Sb=sheep blood, Rb=rabbit blood,Hb=horse blood, Cb=chicken blood, Pb=pork blood, Tb=turkey blood,NSB1=nonspecific binding 1 (antibody only), NSB2=nonspecific binding 2(antigen only), Blk=blank (no antibody, no antigen)

FIG. 12 is a graph depicting the effect of heat treatment on the speciesspecificity of antibody 6F10 when used in an ELISA with soluble proteinsamples from autoclaved, cooked and raw meat as more fully described inExamples 2 and 3. Bb=bovine blood, Sb=sheep blood, Rb=rabbit blood,Hb=horse blood, Cb=chicken blood, Pb=pork blood, Tb=turkey blood,NSB1=nonspecific binding 1 (antibody only), NSB2=nonspecific binding 2(antigen only), Blk=blank (no antibody, no antigen)

FIG. 13 is a graph depicting the detection of autoclaved bovine blood inautoclaved porcine blood by antibodies 6G12 (capture antibody) and 3D6(biotin conjugated detection antibody) when used in a sandwich ELISA asmore fully described in Examples 2 and 3.

FIG. 14 is a graph depicting the detection of spray-dried bovine plasmain spray-dried porcine plasma by antibodies 6G12 (capture antibody) and3D6 (biotin conjugated detection antibody) when used in a sandwich ELISAas more fully described in Examples 2 and 3.

FIG. 15 is a graph depicting the detection of bovine blood in cookedground beef (v/v) by antibodies 6G12 (capture antibody) and 3D6 (biotinconjugated detection antibody) when used in a sandwich ELISA as morefully described in Examples 2 and 3.

FIG. 16 is a graph depicting the detection of bovine blood in raw groundbeef (v/v) by antibodies 6G12 (capture antibody) and 3D6 (biotinconjugated detection antibody) when used in a sandwich ELISA as morefully described in Examples 2 and 3.

FIG. 17 is a graph depicting the detection of bovine blood in cookedground beef (v/w) by antibodies 6G12 (capture antibody) and 3D6 (biotinconjugated detection antibody) when used in a sandwich ELISA as morefully described in Examples 2 and 3.

FIG. 18 is a graph depicting the detection of bovine blood in raw groundbeef (v/w) by antibodies 6G12 (capture antibody) and 3D6 (biotinconjugated detection antibody) when used in a sandwich ELISA as morefully described in Examples 2 and 3.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Advantageously, immunoglobulin peptides have been developed whichspecifically bind antigens in bovine blood. These peptides may be usedin assays for detecting bovine blood in food, animal feed, or othermaterials, and to identify bovine species in forensic, biological, oragricultural sciences. Advantageously, the immunoglobulin peptides arespecific for bovine blood antigens even after a heat treatment of theantigens. For example, monoclonal antibodies of the present inventioncan detect bovine blood in a sample even after the sample has beensterilized to 121° C. for 15 minutes. Thus, the ability of theseimmunoglobulin peptides to react with raw, cooked and autoclaved bovineblood indicates that the antigen is thermostable, allowing for the useof these peptides in a variety of the different assays.

The immunoglobulin peptides of the present invention are highly specificfor bovine blood. For example, the immunoglobulin peptides detect bovineblood in a sample, wherein the sample contains less than 3 wt. % bovineblood or tissue. In one embodiment, the immunoglobulin peptides detectbovine blood in a sample, wherein the sample contains less than 2 wt. %bovine blood or tissue. Preferably, the immunoglobulin peptides detectbovine blood in a sample, wherein the sample contains less than 1 wt. %bovine blood or tissue. In one embodiment, the immunoglobulin peptidesexhibit some cross-reactivity with blood samples of other mammalianspecies, such as ovine, equine or porcine. The cross-reactivity may bedesirable, e.g. in immunoassays with broad specificity for screening anumber of targeted materials with a single test. By way of example, ablood bovine specific antibody, which is cross-reactive with ovine bloodprotein may find application in assaying whether an animal feed samplecontains any blood proteins of ruminant origin. As can be seen fromTable 1, three antibodies (1B4, 2B11, 3D6) of the present inventionexhibit reactivity to blood proteins from two or more mammalian speciesof bovine, ovine, porcine, equine or rabbit origin, whereas fourantibodies (1H9, 6F10, 6G12 and 7F6) react to blood proteins of ruminantspecies (bovine, ovine). In another embodiment, the immunoglobulinpeptides advantageously exhibit little or no cross-reactivity with otherbovine tissues or blood proteins from other animal species. For example,antibody 6E1 only recognizes bovine blood proteins.

Advantageously, the immunoglobulin peptides detect bovine blood evenafter heating or autoclaving treatment. By way of example,immunoglobulin peptides, such as for example, monoclonal antibodies, ofthe present invention detect bovine blood after heating a samplecontaining bovine blood to 100° C. for 15 minutes or even autoclaving itat 121° C. for 15 minutes.

Capture Assay

A capture assay of the present invention employs an immunoglobulinpeptide to bind a bovine blood antigen from the sample. The assays canbe conducted using any procedure selected from the variety of standardassay protocols generally known in the art. In general, the assay isconstructed so as to rely on the interaction of the immunoglobulinpeptide, bovine blood antigen, and a label (or reporter molecule). Inone embodiment, the assay detects the formation of a complex between theimmunoglobulin peptide and bovine blood antigen indirectly; for example,a competitive binding assay may be used in which label is bound to acomposition also having affinity for the immunoglobulin peptide. Inanother embodiment, the assay detects the formation of a complex betweenthe immunoglobulin peptide and bovine blood antigen directly; forexample, a sandwich assay may be employed in which an immunoglobulinpeptide capture agent is bound to the carrier, and a labeled bindingagent, also having affinity for the bovine blood antigen is used toconfirm the binding of antigen by the capture immunoglobulin peptide.The specific design of the assay protocol is open to a wide variety ofchoice, and several clinical assay devices and protocols are availablein the art. In addition, the reaction can be quantitized by comparingagainst a standard curve derived from a known amount(s) of non-taggedmolecules in both indirect and direct detection methodologies.

In one embodiment, an indirect capture assay involves immobilizing animmunoglobulin peptide on a carrier (e.g., solid support or substrate),contacting the coated carrier with the sample, reacting the remainder ofbinding sites on the carrier with a labeled binding agent specific forthe immunoglobulin peptide, and detecting the label. The more bovineblood antigen in the sample, the less tagged binding agent can attach toa given amount of the immunoglobulin peptide on the carrier (the labeledbinding agent is usually supplied in saturation compared to the amountof capture agent).

In one embodiment, a direct capture assay involves immobilizing acapture immunoglobulin peptide on a carrier, contacting the coatedcarrier with the sample, adding labeled immunoglobulin peptide (whichmay be the same as the capture immunoglobulin peptide or another bindingagent having affinity for bovine blood antigens), and detecting thelabel. For direct detection assays, the more thermostable bovine bloodantigen in the sample, the more labeled immunoglobulin peptide canattach to the thermostable bovine blood antigen which is in turn boundto the immunoglobulin peptide on the carrier (the labeled binding agentis usually supplied in saturation). For example, the sample, or adilution thereof, is applied to the immunoglobulin peptide-coatedcarrier under conditions in which the immunoglobulin peptide captureagent binds molecules that display the antigen of interest. The volumeof sample is such that the amount of immobilized immunoglobulin peptideon the carrier is in excess to the expected amount of bovine bloodantigen in the sample. Suitable conditions are, for example, incubationof the potential bovine blood containing sample for about two to threehours at about 37° C. After allowing sufficient time for binding ofthermostable bovine blood antigen to the immunoglobulin peptide, theremaining sample is then washed away.

In an alternative embodiment, a direct capture assay may involveimmobilizing the sample on a carrier and contacting the coated carrierwith labeled immunoglobulin peptide having specificity for bovine bloodantigen. The presence or amount of labeled immunoglobulin peptide boundto the coated carrier may then be correlated directly with the presenceor even amount of bovine blood antigen on the coated carrier.

The capture assays of the present invention can thus be used to detectbovine blood in a sample of food for human or domesticated animalconsumption. In one embodiment, a food sample is any foodstuff used forhuman consumption such as, for example, fresh or canned meat, bloodsausage, soups, bakery products, such as breads and cakes, crackers,butter, wines, and cheeses. Alternatively, a food sample or ingredientfor animal feed for a domesticated animal may be assayed for thepresence (or even amount) of bovine blood, including spray dried blood,bovine plasma, and tissues or substances containing the same; the feedor ingredient may be intended for use, for example, with bovine, swine,and poultry.

Carrier

In general, the carrier may be any of a wide range of suitablesubstrates onto which the immunoglobulin peptide capture agent or bovineblood antigens will attach, usually by electrostatic forces. The carriercan be, for example, plastic or glass material in the form of a tray,bead, or tube, or the carrier can be a suitable membrane of nylon ornitrocellulose. Preferably, the carrier is a plastic microtiter well.Immobilization onto the carrier can occur, for example, by incubatingthe immunoglobulin peptide capture agent or sample in the microtiterwell overnight at about 4° C.

After immobilization, excess immunoglobulin peptide capture agent orsample is removed, and the carrier is usually blocked with a blockingbuffer. For example, the carrier can be blocked with a BSA/PBS buffersolution containing sodium azide for a period of time from about 2 hoursto overnight in a humid atmosphere at room temperature. After blocking,the carrier can be washed with a suitable buffer.

Capture Agent

An immunoglobulin peptide capture agent coating a solid phase materialpreferably binds a sufficient quantity of bovine blood antigen, within arelatively short period of time (approximately several minutes), andretain the captured antigen during subsequent washing and detection oflabeled agents. The density of the immunoglobulin peptide on the carriercan be, for example, from about 1 μg/ml to about 50 μg/ml, and moregenerally around 20 μg/ml. The amount of immunoglobulin peptide captureagent immobilized on the carrier ideally is in excess of the expectedamount of thermostable bovine blood antigen in the sample. Calculatingthe amount of immunoglobulin peptide to be immobilized on the carrier asa function of the expected concentration of thermostable bovine bloodantigen in the sample and the volume of sample delivered is well withinthe skill in the art.

Immunoglobulin peptide capture agents include, for example, polyclonalantibodies, monoclonal antibodies, and antibody fragments such asproteolytically cleaved antibody fragments and single chain Fv antibodyfragments, as further discussed below. In a preferred embodiment, theimmunoglobulin peptide is a monoclonal antibody.

Labeled Binding Agent

After a complex of the bovine blood antigen and the captureimmunoglobulin peptide is formed, the carrier is combined with a labeledbinding agent. The target of the labeled binding agent will depend uponwhether indirect or direct detection means are employed.

In indirect detection assays, the resulting bovine bloodantigen-immunoglobulin peptide complex is further reacted with a bindingagent that has affinity for the immunoglobulin peptide, where thebinding agent is attached to an easily assayable tag. The binding agentpreferably binds with high affinity to immobilized immunoglobulinpeptide capture agent not bound by thermostable bovine blood antigenfrom the sample but does not bind to immobilized immunoglobulin peptidecapture agent already bound by thermostable bovine blood antigen fromthe sample.

In direct detection assays, the resulting thermostable bovine bloodantigen-immunoglobulin peptide complex is further reacted with a bindingagent that has affinity for the thermostable bovine blood antigen, wherethe binding agent is attached to an easily assayable tag. Directdetection binding agents preferably include an immunoglobulin peptidethat displays binding specificity for the thermostable bovine bloodantigen, wherein the labeled immunoglobulin peptide is the sameimmunoglobulin peptide as that used as the capture agent or is a secondimmunoglobulin peptide also having affinity for the thermostable bovineblood antigen.

Immunoglobulin peptides can be used as capture agents and/or bindingagents. Immunoglobulin peptides include, for example, polyclonalantibodies, monoclonal antibodies, and antibody fragments.Immunoglobulin peptides used as capture agents are immobilized on asubstrate surface, as described above. Immunoglobulin peptides used asbinding agents have easily assayed labels or tags affixed, as describedabove.

The following describes generation of immunoglobulin peptides,specifically bovine blood antigen antibodies, via methods that can beused by those skilled in the art to make other suitable immunoglobulinpeptides having similar affinity and specificity which are functionallyequivalent to those used in the Examples.

Hybridoma technology permits one to explore the entire antibodyproducing B lymphocyte repertoire of the immune system and to selectunique antibody producing cells that produce antibodies having uniquebinding characteristics. The production of monoclonal antibodies can bemore controlled than production of polyclonal antisera since polyclonalantisera contain numerous antibody populations each having varyingspecificity and sensitivity characteristics that are the products ofnumerous responding B cell clones. MAb reagents are also homogeneouswith a defined specificity. The use and appropriate selection ofhybridoma cell lines provides MAb reagents that offer unique performancecharacteristics to the test system and consistency of the methods thatutilize them.

MAbs can be screened or tested for specificity using any of a variety ofstandard techniques, including Western Blotting (Koren, E. et al. (1986)Biochim. Biophys. Acta 876:91-100) and enzyme-linked immunosorbent assay(ELISA) (Koren, E. et al. (1986) Biochim. Biophys. Acta 876:91-100).

Methods for the preparation of the antibodies of the present inventionare generally known in the art. See, for example, Antibodies, ALaboratory Manual, Ed. Harlow & David Lane (eds.) Cold Spring HarborLaboratory, N.Y. (1988), as well as the references cited therein.Standard reference works setting forth the general principles ofimmunology include: Klein, J. Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, N.Y. (1982); Dennett, R. et al.Monoclonal Antibodies, Hybridoma: A New Dimension In Biological AnalysesPlenum Press, N.Y. (1980); and Campbell, A. “Monoclonal AntibodyTechnology,” Laboratory Techniques In Biochemistry And MolecularBiology, Vol. 13, Burdon et al. (eds.), Elsevier, Amsterdam (1984). Seealso, U.S. Pat. Nos. 4,609,893; 4,713,325; 4,714,681; 4,716,111;4,716,117; and 4,720,459.

Any method can be used to generate antibodies, including but not limitedto methods that elicit production of monoclonal antibodies. In oneembodiment, animals such as mice are inoculated with the immunogen inadjuvant, and spleen cells are harvested and mixed with a myeloma cellline, such as P3X63Ag8,653. The cells are induced to fuse by theaddition of polyethylene glycol. Hybridomas are chemically selected byplating the cells in a selection medium containing hypoxanthine,aminopterin and thymidine (HAT). Hybridomas are subsequently screenedfor the ability to produce anti-rendered animal byproduct monoclonalantibodies. Hybridomas producing antibodies are cloned, expanded andstored frozen for future production.

In another embodiment, antibodies are generated by immunizing an animalwith an immunogenic amount of the antigen emulsified in an adjuvant suchas Freund's complete adjuvant, administered over a period of weeks inintervals ranging between two weeks and 6 weeks. In a some embodiments,the method includes a first immunization in Freund's complete adjuvantand subsequent immunizations in Freund's incomplete adjuvant (atbiweekly to monthly intervals thereafter) then isolating the antibodiesfrom the serum, or fusing spleen from the animal cells to myeloma cellsto make hybridomas which express the antibodies in culture. In someembodiments, test bleeds are taken at fourteen day intervals between thesecond and third immunizations and production bleeds at monthlyintervals thereafter.

In one embodiment, the immunoglobulin peptides of the present inventionare monoclonal antibodies of the IgG class. Exemplary IgG monoclonalantibodies include, for example, monoclonal antibodies selected from thegroup consisting of 1B4.B12.E12 (1B4), 1H9.B5.D9 (1H9), 2B11.F3.B9(2B11), 3D6.C7.G9 (3D6), 6E1.E5.D2.B9 (6E1), 6F10.A10.B8.A11 (6F10),6G12.G1.A9 (6G12) and 7F6.C1.H11 (7F6), produced by hybridoma cell linesdeposited as ATCC Nos. PTA-9870, PTA-9869, PTA-9868, PTA-9867, PTA-9982,PTA-9983, PTA-9985 and PTA-9984, respectively. Biological deposits ofhybridomas assigned ATCC accession numbers PTA-9867, PTA-9868, PTA-9869,and PTA-9870 were received by the American Type Culture Collection(ATCC, Manassas, Va.) on Mar. 5, 2009 and deposited as of Apr. 3, 2009.Biological deposits of hybridomas assigned ATCC accession numbersPTA-9982, PTA-9983, PTA-9984, and PTA-9985 were received by the AmericanType Culture Collection (ATCC, Manassas, Va.) on Apr. 30, 2009 anddeposited as of Jun. 4, 2009. In one preferred embodiment, for example,the monoclonal antibodies are 6E1, 6G12, 6F10, 1H9, 7F6, or acombination thereof. In another embodiment, the monoclonal antibody is6E1.

It may be desirable to produce and use functional fragments of amonoclonal antibody for a particular application. The well-known basicstructure of a typical IgG molecule is a symmetrical tetrameric Y-shapedmolecule of approximately 150,000 to 200,000 daltons consisting of twoidentical light polypeptide chains (containing about 220 amino acids)and two identical heavy polypeptide chains (containing about 440 aminoacids). Heavy chains are linked to one another through at least onedisulfide bond. Each light chain is linked to a contiguous heavy chainby a disulfide linkage. An antigen-binding site or domain is located ineach arm of the Y-shaped antibody molecule and is formed between theamino terminal regions of each pair of disulfide linked light and heavychains. These amino terminal regions of the light and heavy chainsconsist of approximately their first 110 amino terminal amino acids andare known as the variable regions of the light and heavy chains. Inaddition, within the variable regions of the light and heavy chainsthere are hypervariable regions which contain stretches of amino acidsequences, known as complementarity determining regions (CDRs). CDRs areresponsible for the antibody's specificity for one particular site on anantigen molecule called an epitope. Thus, the typical IgG molecule isdivalent in that it can bind two antigen molecules because eachantigen-binding site is able to bind the specific epitope of eachantigen molecule. The carboxy terminal regions of light and heavy chainsare similar or identical to those of other antibody molecules and arecalled constant regions. The amino acid sequence of the constant regionof the heavy chains of a particular antibody defines what class ofantibody it is, for example, IgG, IgD, IgE, IgA or IgM. Some classes ofantibodies contain two or more identical antibodies associated with eachother in multivalent antigen-binding arrangements.

Proteolytic cleavage of a typical IgG molecule with papain is known toproduce two separate antigen binding fragments called Fab fragmentswhich contain an intact light chain linked to an amino terminal portionof the contiguous heavy chain via by disulfide linkage. The remainingportion of the papain-digested immunoglobulin molecule is known as theFc fragment and consists of the carboxy terminal portions of theantibody left intact and linked together via disulfide bonds. If anantibody is digested with pepsin, a fragment known as an F(ab′)2fragment is produced which lacks the Fc region but contains bothantigen-binding domains held together by disulfide bonds betweencontiguous light and heavy chains (as Fab fragments) and also disulfidelinkages between the remaining portions of the contiguous heavy chains(Handbook of Experimental Immunology. Vol 1: Immunochemistry, Weir, D.M., Editor, Blackwell Scientific Publications, Oxford (1986)).

Fab and F(ab′)₂ fragments of MAbs that bind thermostable bovine bloodantigen can be used in place of whole MAbs in methods for detecting orquantifying thermostable bovine blood antigen in samples. Because Faband F(ab′)₂ fragments are smaller than intact antibody molecules, moreantigen-binding domains can be immobilized per unit area of a solidsupport than when whole antibody molecules are used. As explained below,rapid, easy, and reliable assay systems can be made in which antibodiesor antibody fragment that specifically bind thermostable bovine bloodantigen are immobilized on solid phase materials.

Recombinant DNA methods have been developed which permit the productionand selection of recombinant immunoglobulin peptides which are singlechain antigen-binding polypeptides known as single chain Fv fragments(ScFvs or ScFv antibodies). ScFvs bind a specific epitope of interestand can be produced using any of a variety of recombinant bacterialphage-based methods, for example as described in Lowman et al. (1991)Biochemistry, 30: 10832-10838; Clackson et al. (1991) Nature 352:624-628; and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382. These methods are usually based on producing geneticallyaltered filamentous phage, such as recombinant M13 or fd phages, whichdisplay on the surface of the phage particle a recombinant fusionprotein containing the antigen-binding ScFv antibody as the aminoterminal region of the fusion protein and the minor phage coat proteing3p as the carboxy terminal region of the fusion protein. Suchrecombinant phages can be readily grown and isolated using well-knownphage methods. Furthermore, the intact phage particles can usually bescreened directly for the presence (display) of an antigen-binding ScFvon their surface without the necessity of isolating the ScFv away fromthe phage particle.

To produce an ScFv, standard reverse transcriptase protocols are used tofirst produce cDNA from mRNA isolated from a hybridoma that produces anMAb for bovine blood antigen. The cDNA molecules encoding the variableregions of the heavy and light chains of the MAb can then be amplifiedby standard polymerase chain reaction (PCR) methodology using a set ofprimers for mouse immunoglobulin heavy and light variable regions(Clackson (1991) Nature 352: 624-628). The amplified cDNAs encoding MAbheavy and light chain variable regions are then linked together with alinker oligonucleotide in order to generate a recombinant ScFv DNAmolecule. The ScFv DNA is ligated into a filamentous phage plasmiddesigned to fuse the amplified cDNA sequences into the 5′ region of thephage gene encoding the minor coat protein called g3p. Escherichia colibacterial cells are then transformed with the recombinant phageplasmids, and filamentous phage grown and harvested. The desiredrecombinant phages display antigen-binding domains fused to the aminoterminal region of the minor coat protein. Such “display phages” canthen be passed over immobilized antigen, for example, using the methodknown as “panning”, see Parmley and Smith (1989) Adv. Exp. Med. Biol.251: 215-218; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382, to absorb those phage particles containing ScFv antibodyproteins that are capable of binding antigen. The antigen-binding phageparticles can then be amplified by standard phage infection methods, andthe amplified recombinant phage population again selected forantigen-binding ability. Such successive rounds of selection forantigen-binding ability, followed by amplification, select for enhancedantigen-binding ability in the ScFvs displayed on recombinant phages.Selection for increased antigen-binding ability may be made by adjustingthe conditions under which binding takes place to require a tighterbinding activity. Another method to select for enhanced antigen-bindingactivity is to alter nucleotide sequences within the cDNA encoding thebinding domain of the ScFv and subject recombinant phage populations tosuccessive rounds of selection for antigen-binding activity andamplification (see Lowman et al. (1991) Biochemistry 30: 10832-10838;and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382).

Once a ScFv is selected, the recombinant thermostable bovine bloodantigen antibody can be produced in a free form using an appropriatevector in conjunction with E. coli strain HB2151. These bacteriaactually secrete ScFv in a soluble form, free of phage components(Hoogenboom et al. (1991) Nucl. Acids Res. 19: 4133-4137). Thepurification of soluble ScFv from the HB2151 bacteria culture medium canbe accomplished by affinity chromatography using antigen moleculesimmobilized on a solid support such as AFFIGEL™ (BioRad, Hercules,Calif.).

Other developments in the recombinant antibody technology demonstratepossibilities for further improvements such as increased avidity ofbinding by polymerization of ScFvs into dimers and tetramers (seeHolliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448).

Because ScFvs are even smaller molecules than Fab or F(ab′)2 fragments,they can be used to attain even higher densities of antigen bindingsites per unit of surface area when immobilized on a solid supportmaterial than possible using whole antibodies, F(ab′)2, or Fabfragments. Furthermore, recombinant antibody technology offers a morestable genetic source of antibodies, as compared with hybridomas.Recombinant antibodies can also be produced more quickly andeconomically using standard bacterial phage production methods.

In one embodiment of the present invention, therefore, theimmunoglobulin peptide is a monoclonal antibody fragment which retainsthe ability to bind antigens from bovine blood. In one embodiment, thesefragments include Fab and Fab2 portions of 1B4, 1H9, 2B11, 3D6, 6E1,6F10, 6G12 and 7F6. In another embodiment, Fab and Fab2 fragments arefrom 6E1, 1H9, 6G12, 6F10, and 7F6 antibodies. Such fragments aretypically produced by proteolytic cleavage, such as papain or pepsin.Alternatively, antigen-binding fragments can be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

Detection

Regardless of whether the assay is a direct or indirect method, a label(reporter molecule) is bound to a molecule having affinity for (i) thebovine blood antigen or (ii) an immunoglobulin peptide (theimmunoglobulin peptide, in turn, having affinity for the bovine bloodantigen). Thus, for example, the assay may comprise (i) a sandwich assayin which a capture immunoglobulin peptide is bound to the carrier, thecapture immunoglobulin peptide binds bovine blood antigen in the sample,and the captured bovine blood antigen then binds a labeledimmunoglobulin peptide, (ii) a direct assay in which sample is bound tothe carrier and is exposed to labeled immunoglobulin peptide, or (iii)an indirect assay in which a capture immunoglobulin peptide is bound tothe carrier, the capture immunoglobulin peptide binds bovine bloodantigen in the sample, and any unbound capture immunoglobulin peptidethen binds a labeled composition having affinity for the captureimmunoglobulin peptide. For such uses, a wide range of labels anddetection methods may be employed.

Detection methodology will depend upon the identity of the assayable tagon the binding agent, as commonly understood in the art. Kits fordetection of tagged binding agents in the capture immunoassay describedabove are commercially available. Detection procedures include Westernblots, enzyme-linked immunosorbent assays, radioimmunoassays,competition immunoassays, dual antibody sandwich assays,immunohistochemical staining assays, agglutination assays, andfluorescent immunoassays.

The assayable tag may be detectable directly or may bind to a reporterfor which it has specificity. The assayable tag attached to the bindingagent can be, for example, an enzyme, a coenzyme, an enzyme substrate,an enzyme co-factor, an enzyme inhibitor, a radionuclide, a chromogen, afluorescer, a chemoluminescer, a free radical, or a dye. Alternatively,detection can be mediated by reporter reagents such as fluorescentavidins, streptavidins or other biotin-binding proteins orenzyme-conjugated streptavidins plus a fluorogenic, chromogenic, orchemiluminescent substrate.

In one embodiment, the tag is biotin, which is then recognized by avidinor streptavidin conjugated to a reporter, such as the enzyme horseradishperoxidase. For example, the tagged binding agent can be biotinylatedASF or biotinylated isolectin B4 from Vicia villosa lectin (VVLB4).Biotin is typically conjugated to proteins via primary amines (i.e.,lysines). Usually, between three and six biotin molecules are conjugatedto each binding agent molecule. The avidin homolog streptavidin, whichis secreted by Streptomyces avidinii, is preferred as a linking agentbecause of its particularly high affinity for biotin.

A number of fluorescent compounds such as fluorescein isothiocyanate,europium, lucifer yellow, rhodamine B isothiocyanate (Wood (1991) In:Principles and Practice of Immunoassay, Stockton Press, New York, pp.365-392) can be used to label binding agents. In conjunction with theknown techniques for separation of antibody-antigen complexes, thesefluorophores can be used to quantify thermostable bovine blood antigenin samples. The same applies to chemiluminescent immunoassay in whichcase either thermostable bovine blood antigen antibody can be labeledwith isoluminol or acridinium esters (Krodel (1991) In: Bioluminescenceand Chemiluminescence: Current Status. John Wiley and Sons Inc. NewYork, pp 107-110; Weeks (1983) Clin. Chem. 29:1480-1483).Radioimmunoassay (Kashyap, M. L. et al., J. Clin. Invest. 60:171-180(1977)) is another technique in which thermostable bovine blood antigenantibodies can be used after coupling with a radioactive isotope such as¹²⁵1. Some of these immunoassays can be easily automated by the use ofappropriate instruments such as the IMX™ (Abbott, Irving, Tex.) for afluorescent immunoassay and Ciba Corning ACS 18O™ (Ciba Corning,Medfield, Mass.) for a chemiluminescent immunoassay. Kits for detectionof tagged binding agents in ELISA procedures are commercially available.

In one embodiment, a streptavidin/peroxidase complex is used to assaythe amount of biotin tag. The activity of the peroxidase enzyme linkedto the streptavidin can then be detected through the addition of aperoxidase substrate. An example of a peroxidase substrate is2,2′-Azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) (ABTS).

Solutions with known amounts of bovine blood antigen can be used in thegeneration of standard curves.

In some embodiments, non-radioactive labels are attached by indirectmeans. In some embodiments, a ligand molecule (e.g., biotin) iscovalently bound to the molecule. The ligand then binds to ananti-ligand (e.g., streptavidin) molecule which is either inherentlydetectable or covalently bound to a signal system, such as a detectableenzyme, a fluorescent compound, or a chemiluminescent compound. Anyligands and anti-ligands that will function can be used. In someembodiments in which the ligand has a natural antiligand, for example,biotin, thyroxine, and cortisol, it is used in conjunction with thelabeled, naturally occurring anti-ligands. Alternatively, any haptenicor antigenic compound can be used in combination with an antibody.

In some embodiments, the immunoglobulin peptides are conjugated directlyto signal generating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol.

As previously noted, detection of labels may occur by a range ofmethods. Examples of known methods include, but are not limited toimmunoblotting, western blot analysis, gel-mobility shift assays,fluorescent in situ hybridization analysis (FISH), tracking ofradioactive or bioluminescent markers, nuclear magnetic resonance,electron paramagnetic resonance, stopped-flow spectroscopy, columnchromatography, capillary electrophoresis, or other methods which tracka molecule based upon an alteration in size and/or charge. Any means maybe used to detect labels. Thus, for example, where the label is aradioactive label, examples of means for detection include ascintillation counter or photographic film as in autoradiography. Wherethe label is a fluorescent label, it may be detected, for example, byexciting the fluorochrome with the appropriate wavelength of light anddetecting the resulting fluorescence, e.g., by microscopy, visualinspection, via photographic film, by the use of electronic detectorssuch as charge coupled devices (CCDs) or photomultipliers and the like.Similarly, enzymatic labels are detected, for example, by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detected,for example, by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears dark purple,while various conjugated beads appear the color of the bead.

In another aspect, the present invention provides a method for detectingbovine blood in a food sample or an animal feed sample. The methodcomprises the steps of coating a solid surface with a first monoclonalantibody selected from the group consisting of 1B4, 1H9, 2B11, 3D6, 6E1,6F10, 6G12 and 7F6; contacting the first monoclonal antibody with thefood sample or the animal feed sample; contacting the food sample or theanimal feed sample with a second monoclonal antibody selected from thegroup consisting of 1B4, 1H9, 2B11, 3D6, 6E1, 6F10, 6G12 and 7F6,wherein the second monoclonal antibody is different from the firstmonoclonal antibody and is conjugated to a detectable marker; andobtaining a detectable signal generated by said marker. This method canbe performed by, for example, a sandwich immunoassay.

The present invention also provides an alternative method for detectingthe presence of bovine blood in a food sample or an animal feed sample,wherein the method comprises coating a solid surface with the foodsample or the animal feed sample; contacting the food sample or theanimal feed sample with a monoclonal antibody selected from the groupconsisting of 1B4, 1H9, 2B11, 3D6, 6E1, 6F10, 6G12 and 7F6; contactingthe monoclonal antibody with a detection antibody capable of bindingsaid monoclonal antibody, wherein the detection antibody is conjugatedto a detectable marker; and obtaining a detectable signal generated bysaid detectable marker. This method can be performed, for example, bynon-competitive indirect ELISA.

The sandwich ELISA is used to determine the antigen concentration inunknown samples. For purposes of the present invention, the sample is afood sample or an animal feed sample. In one embodiment, a food sampleis selected from meats. In another embodiment, a food sample is selectedfrom ground meats. In another embodiment, the food sample is selectedfrom butter and cheeses. In another embodiment, the food sample isselected from breads, cakes, crackers and other bakery products. In yetanother embodiment, a food sample is selected from wines. An animal feedsample is selected from, for example, meat, bone meal, and proteinenriched fodder containing bovine components, such as for example, spraydried bovine blood or plasma, or meat and bone meal components. In oneembodiment, the animal feed sample is selected from meat and bone meal.In still another embodiment, the animal feed sample is fodder enrichedwith meat and bone meal or a bovine component.

The preparation of a food sample and an animal feed sample is the samefor purposes of ELISA. Briefly, a food sample or an animal feed sampleis cooked or autoclaved, e.g., by heating in a boiling water bath forabout 15 minutes or by autoclaving, for example, for 15 minutes at 121°C. at 1.2 bars. Next, the cooked and autoclaved samples are mashed intofine particles and the extraction buffer is added. The samples are thenhomogenized and centrifuged, and the supernatants are used for ELISA,following determination of protein concentration. A complete protocolfor preparing food or animal feed samples is described in Example 2.

The sandwich ELISA is fast and accurate, and if a purified antigenstandard is available, the assay can determine the absolute amount ofantigen in an unknown sample. The sandwich ELISA requires two antibodiesthat bind to epitopes that do not overlap on the antigen. This can beaccomplished with either two monoclonal antibodies that recognizediscrete sites or one batch of affinity-purified polyclonal antibodies.In one embodiment, the two monoclonal antibodies used in the ELISA areselected from the group consisting of 1B4, 1H9, 2B11, 3D6, 6E1, 6F10,6G12 and 7F6. In another embodiment, the pair of monoclonal antibodiesis 3D6 and 7F6. In another embodiment, the pair of monoclonal antibodiesis 1B4 and 6F10. In another embodiment, the pair of monoclonalantibodies is 6G12 and 7F6. Additional antibody combinations can bedetermined based on antibody specificities as shown in Table 2. Withrespect to the labeling of a monoclonal antibody for purposes of signaldetection, any label discussed above can be used. In one embodiment, thelabel is selected from biotin, horseradish peroxidase, fluoresceinisothiocyanate, gold, and rhodamine B isothiocyanate.

To utilize this assay, one antibody (the “capture” antibody) is purifiedand bound to a solid phase typically attached to the bottom of a platewell. Antigen is then added and allowed to complex with the boundantibody. Unbound products are then removed with a wash, and a labeledsecond antibody (the “detection” antibody) is allowed to bind to theantigen, thus completing the “sandwich.” The assay is then quantitatedby measuring the amount of labeled second antibody bound to the matrixthrough the use of a colorimetric substrate. Major advantages of thistechnique are that the antigen does not need to be purified prior to useand that these assays are very specific. However, one disadvantage isthat not all antibodies can be used. Monoclonal antibody combinationsmust be qualified as “matched pairs,” meaning that they can recognizeseparate epitopes on the antigen so they do not hinder each other'sbinding.

A general protocol for the sandwich ELISA method is described below.Before the assay, both antibody preparations should be purified and onemust be labeled. For most applications, a polyvinylchloride (PVC)microtiter plate is used. The unlabeled antibody is bound to the bottomof each well by adding approximately 50 μL of antibody solution to eachwell (e.g. 20 μg/mL in PBS). The amount of antibody used will depend onthe individual assay. The plate is incubated overnight at 4° C. to allowcomplete binding. Next, the wells are washed, for example, two time,with PBS. The remaining sites for protein binding on the microtiterplate must be saturated by incubating with blocking buffer. This isgenerally done by filling the wells to the top with 1% BSA/PBS with0.02% sodium azide, following which the plate is incubated for 2 hoursto overnight in a humid atmosphere at room temperature. It is of notethat sodium azide is not included in buffers or wash solutions if anHRP-labeled antibody will be used for detection. The wells are thenwashed, for example, two times, with PBS.

The antigen solution (generally 50 μL) is added to the wells. Theantigen solution should generally be titrated. All dilutions should bedone in the blocking buffer (1% BSA/PBS), and the plate is generallyincubated for at least about 2 hours at room temperature in a humidatmosphere.

The plate is again washed with PBS, and the labeled second antibody isadded. For accurate quantification, the second antibody generally isused in excess. All dilutions should be done in the blocking buffer. Theplate is again incubated for about 1-2 hours or more at room temperaturein a humid atmosphere and washed with several changes of PBS. Thesubstrate is next added, and after the appropriate incubation time haselapsed, optical densities at target wavelengths can be measured on anELISA plate reader.

In an alternative embodiment, when two “matched pair” antibodies are notavailable, another option is the competitive ELISA. Because of theprobability for steric hindrance occurring when two antibodies attemptto bind to a small molecule at the same time, a competitive inhibitionassay can be used in such a case. An advantage to the competitive ELISAis that non-purified primary antibodies may be used. There are severaldifferent configurations for competitive ELISAs, which are known to oneof ordinary skill in the art. Below is an example for one suchconfiguration.

In order to utilize a competitive ELISA, one reagent must be conjugatedto a detection label, such as horseradish peroxidase. This enzyme may belinked to either the immunogen or the primary antibody. The protocolbelow uses a labeled immunogen as the competitor.

Briefly, an unlabeled purified primary antibody is coated onto the wellsof a 96 well microtiter plate. This primary antibody is then incubatedwith unlabeled standards and unknowns. After this reaction is allowed togo to equilibrium, conjugated immunogen is added. This conjugate willbind to the primary antibody wherever its binding sites are not alreadyoccupied by unlabeled immunogen. Thus, the more immunogen in the sampleor standard, the lower the amount of conjugated immunogen bound. Theplate is then developed with substrate and color change is measured.

In another embodiment, a competitive ELISA is performed by coating theplate with immunogen, such as autoclaved bovine blood, adding anenzyme-conjugated monoclonal antibody selected from 1B4, 1H9, 2B11, 3D6,6E1, 6F10, 6G12 and 7F6 together with an unknown sample, wherein theantigen in the unknown sample competes with the immobilized immunogen,and developing the plate.

An exemplary protocol for a competitive ELISA method is described below.

A diluted primary antibody (capture) is added to each well (generally 50μL). The appropriate dilution should generally be determined prior totesting samples. The plate is then incubated for 4 hours at roomtemperature or 4° C. overnight. If a purified capture antibody is notavailable, the plate should generally first be coated with a purifiedsecondary antibody directed against the host of the capture antibodyaccording to the following procedure:

The unlabeled secondary antibody may be bound to the bottom of each well(e.g., by adding approximately 50 μL of antibody solution to each well).The plate is incubated overnight at 4° C., and the primary captureantibody is added.

The wells are then washed with PBS (e.g., twice). Next, the wells areincubated with 1% BSA/PBS with 0.02% sodium azide, and incubated for 1-2hours to overnight in a humid atmosphere at room temperature, followingwhich they are washed with PBS. The standards and sample solutions areadded to the wells, wherein all dilutions are generally done in theblocking buffer (1% BSA/PBS with 0.05% Tween-20). Sodium azide shouldgenerally not be used in buffers or wash solutions, if an HRP-Iabeledconjugate is used for detection.

The antigen-conjugate solution (e.g., 50 μL) is added to the wells (theantigen solution should generally be titrated) and incubated for atleast 2 hours at room temperature in a humid atmosphere, following whichthe plate is washed (e.g., four times) with PBS.

The substrate is next added to the plate, incubated for an appropriateamount of time, and optical densities at target wavelengths are measuredon an ELISA reader. It is of note that competitive ELISAs yield aninverse curve, where higher values of antigen in the samples orstandards yield a lower amount of color change.

In a sequential competitive inhibition assay, the sample and conjugatedanalyte are added in steps like a sandwich assay, while in a classiccompetitive inhibition assay, these reagents are incubated together atthe same time.

In a sequential competitive inhibition assay format, a monoclonalantibody is coated onto a 96-well microtiter plate. When the sample isadded, the monoclonal captures free analyte out of the sample. In thenext step, a known amount of analyte labeled with either biotin or HRPis added. The labeled analyte will then also attempt to bind to the MAbadsorbed onto the plate; however, the labeled analyte is inhibited frombinding to the MAb by the presence of previously bound analyte from thesample. This means that the labeled analyte will not be bound by themonoclonal on the plate if the monoclonal has already bound unlabeledanalyte from the sample.

The amount of unlabeled analyte in the sample is inversely proportionalto the signal generated by the labeled analyte. The lower the signal,the more unlabeled analyte there is in the sample. A standard curve canbe constructed using serial dilutions of an unlabeled analyte standard.Subsequent sample values can then be read off the standard curve as isdone in the sandwich ELISA formats.

Both sandwich ELISA and competitive ELISA are frequently used in theart, and a skilled artisan can readily modify the above describedprotocols.

Kits

The capture agent(s), labeled binding agent(s), revealing reagents,and/or standards for the conduct of the various capture immunoassaysdescribed herein may conveniently be supplied as kits which include thenecessary components and instructions for performing the assay.Screening/diagnostic kits typically comprise one or more reagents thatspecifically bind to the target that is to be screened (e.g., ligandsthat specifically bind to thermostable bovine blood antigens). Thereagents can, optionally, be provided with an attached label and/oraffixed to a substrate (e.g. as a component of a protein array), and/orcan be provided in solution. The kits can comprise nucleic acidconstructs (e.g., vectors) that encode one or more such ligands tofacilitate recombinant expression of such. The kits can optionallyinclude one or more buffers, detectable labels or labeled bindingagents, or other reagents as may be useful in a particular assay.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the practice of themethods described herein. Preferred instructional materials describe thedetection of thermostable bovine blood antigen in animal feed or othersamples. While the instructional materials typically comprise written orprinted materials, they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

A preferred kit includes a microtiter plate coated with a thermostablebovine blood antigen immunoglobulin peptide capture agent, standardsolutions for preparation of standard curve, a control for qualitytesting of the analytical run, thermostable bovine blood antigenconjugated to biotin, streptavidinperoxidase enzyme, a substratesolution, a stopping solution, a washing buffer, and an instructionmanual.

In some embodiments, the antibody is collectively assembled in a kitwith conventional immunoassay reagents for detection of bovine blood.The kit may optionally contain a standard for the determination of thepresence of bovine blood in a sample. The kit containing these reagentsprovides for simple, rapid, on site detection of bovine blood in a foodsample or an animal feed sample.

For purposes of the kit, the monoclonal antibodies can be coated onto asolid phase, such as an ELISA microliter plate, later flow strips, adipstick, magnetic beads, and the like, and used as a sensitive reagentto accurately detect bovine blood. This commercial kit form is usefulfor rapid and convenient use by regulatory agencies and the meatindustry. The kit can be formulated to contain antibodies for anon-competitive ELISA, including double-sandwich ELISA assays andindirect ELISA, as well as competitive assays. However, other formatssuch as homogenous enzyme immunoassays may be developed. In oneembodiment, the kit for performing sandwich ELISA contains theantibodies 3D6 and 6g12. In another embodiment, the kit for performing acompetitive ELISA contains at least one of the antibodies 1B4, 1H9,2B11, 3D6, 6E1, 6F10, 6G12 and 7F6. In another embodiment, the kit forperforming a competitive ELISA contains at least one of 6E1, 6F10, 7F6or 1H9.

In some embodiments, at least one monoclonal antibody that is used in akit is labeled. A wide variety of labels and conjugation techniques areknown and are reported extensively in both the scientific and patentliterature. In some embodiments, the antibody is labeled indirectly byreaction with labeled substances that have an affinity for the antibody,such as protein A or G. Any label, detectable group, conjugationtechnique or other method of labeling may be used. Suitable labelsinclude radioactive molecules, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. The detectable group can be any material havinga detectable physical or chemical property. Such detectable labels havebeen well-developed and, in general, any label useful in such methodscan be applied to the present method. Thus, a label is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical, or any other means. Useful labels in thepresent invention include fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,3H, 1251, 35S, 14C, or 32P), enzymes (e.g., LacZ, CAT, horseradishperoxidase, alkaline phosphatase and others, commonly used as detectableenzymes, either in an EIA or in an ELISA), and colorimetric labels suchas colloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads. The label may be coupled directly orindirectly to the desired component of the assay according to methodswell known in the art. As indicated above, a wide variety of labels maybe used, with the choice of label depending on the sensitivity required,ease of conjugation of the compound, stability requirements, availableinstrumentation, and disposal provisions.

Other features, objects and advantages of the present invention will beapparent to those skilled in the art. The explanations and illustrationspresented herein are intended to acquaint others skilled in the art withthe invention, its principles, and its practical application. Thoseskilled in the art may adapt and apply the invention in its numerousforms, as may be best suited to the requirements of a particular use.Accordingly, the specific embodiments of the present invention as setforth are not intended as being exhaustive or limiting of the presentinvention.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

Definitions and Abbreviations

The term “animal byproduct” as used herein means one or more of thoseparts or portions of animals that are typically discarded during theprocessing of animals for the preparation of meat products from animalsfor human consumption. Examples include, but are not limited to bone,connective tissue (e.g., cartilage, tendons, ligaments, and fascia),skin, hair, feathers, beaks, hooves, horns, claws, fat, greaves, blood,certain muscles, and combinations thereof.

The term “antigen” refers to a foreign substance that, when introducedinto the body, can stimulate an immune response.

The term “analyte,” refers to a substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as anantibody), or for which a specific binding member can be prepared.

The term “animal feed” includes any food stuff that is used to feedlivestock, such as cattle, sheep, goats, pigs, chickens, turkey, ducks,etc.

The term “epitope” means an antigenic determinant of a polypeptide orprotein.

The terms “Fab” and “Fab2” refer to antibody fragments obtained bydigestion with papain and pepsin, respectively. Fab contains a singleantigen binding site, whereas Fab2 contains two antigen-binding sites.Fab and Fab2 also include such fragments which are produced byrecombinant DNA technology or synthetic chemistry.

The term “monoclonal antibody” refers to a single type of antibody thatis directed against a specific epitope (antigenic determinant) and isproduced by a single clone of B cells or a single hybridoma cell line.“MAb” is an abbreviation for monoclonal antibody.

The term “polyclonal antibody” refers to a mixture of antibodies activeagainst a specific antigen, each recognizing a different epitope orregion of the antigen.

The term “rendered” as used herein, is defined to have its broadestpossible meaning to include all types of rendering processes in theanimal meat processing and packaging industry, including processes thatinclude a step of physically milling, grounding, or otherwise processinginto particles of small size and heating. The purpose of heating may be,for example, to kill pathogens, to render the material more digestible,to separate fat from non-fat materials, or all of the them. Onenon-limiting example of a rendering temperature is between approximately121° and approximately 138° C., although heating temperatures varydepending on whether the heating is performed under pressure as well asthe duration of heating.

The term “rendered animal byproduct” as used herein shall mean animalbyproduct that has been rendered.

The term “meat and bone meal” (or “MBM”) as used herein refers to a typeof rendered animal byproduct. MBM is made by rendering animal byproductsof the meat packing industry. Examples of commercial providers of MBMinclude, but are not limited to, ConAgra Foods, (Greeley Colo.), DarlingInternational (Irving, Tex.); Excel Corporation (Wichita, Kans.);National By-Products, (Des Moines, Iowa); and Valley Proteins, Inc.,(Winchester, Va.).

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing the scope ofthe invention defined in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure, whileillustrating the invention, are provided as non-limiting examples andare, therefore, not to be taken as limiting the various aspects of theinvention so illustrated.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Production of Monoclonal Antibodies Specific to Bovine BloodThermal Stable Proteins Extraction of Thermal-Stable Soluble Proteins(TSPs) From Whole Animal Blood

Soluble TSPs were extracted from cooked and autoclaved whole bloodsample from each species (bovine, porcine, ovine, equine, rabbit, turkeyand chicken). Twenty mL of each blood sample was dispensed into a 50-mlbeaker to prepare the “autoclaved blood sample.” The beaker was coveredwith aluminum foil and autoclaved for 15 minutes at 121° C. at 1.2 bars.Another beaker containing 20 mL of blood sample from each species washeated in a boiling water bath for 15 minutes to obtain “cooked bloodsample.” Both autoclaved and cooked blood samples from each species werethen mashed into fine particles using a glass rod. An equal volume (20mL) of the extraction buffer (10 mM phosphate buffered saline) was addedto the mashed samples to extract the soluble TSPs and the mixture washomogenized for 2 minutes at 11000 RPM using the ULTRA-TURRAX T25 basichomogenizer (IKA Works Inc., Wilmington, N.C.). The homogenized sampleswere covered with paraffin films and allowed to stand for 2 hours atroom temperature followed by another 2 hours at 4° C. The mixtures werethen transferred into centrifuge tubes and centrifuged (Eppendorf 581ORcentrifuge, Brinkman Instruments Inc., Westbury, N.Y.) at 3220×g for 60minutes at 4° C. The supernatants of cooked and autoclaved blood sampleswere then filtered through Whatman No. 1 filter paper. The proteinconcentration of clear filtrates was determined by protein assay kit II(Bio-Rad, Hercules, Calif.) based on the Bradford dye-binding method.Bovine serum albumin (BSA) was used as the standard in this assay. Allprotein extracts were stored at −20° C. until use.

Production of Monoclonal Antibodies Specific to Bovine TSPs Immunization

The autoclaved bovine blood TSP crude extract was dialyzed in 10 mM PBSfor 24 hours and the dialyzed extract was used as the immunogen. Thispartially purified crude protein extract was used to immunize animalsbecause all individual TSP in the extract could be a potential antigento elicit antibody production. Three female BALB/c mice were immunizedsubcutaneously with 100 μg/mouse of the dialyzed TSPs in phosphatebuffered saline (PBS) emulsified with an equal volume of Freund'scomplete adjuvant. Several boost injections prepared in the same mannerusing Freund's incomplete adjuvant were applied to each mouse at 4-weekintervals. Test sera from mice were collected 8 days after each boostingby tail bleeding. The titer of the sera was determined by indirectELISA. The mouse showing the highest titer was injectedintraperitoneally with 100 μg of marker protein in PBS 4 days prior tofusion.

Hybridoma Procedures

The spleen cells from the immunized mouse were fused with murine myelomacells (P3×63.Ag8.653, ATCC CRL 1580) for hybridoma production. Thegeneral procedures as described by Kohler and Milstein (1975) werefollowed with modifications and the following specific screeningprocedures. Those hybridomas secreting antibodies that react with thetarget antigen were selected, cloned twice by limiting dilution, andexpanded. The initial screening against the antigen (autoclaved bovineTSPs) was performed using indirect ELISA. For a secondary selection, thepositive cells from the initial screening were expanded and thesupernatants were screened for reactivity with the native blood serumproteins, TSPs from the cooked blood samples from bovine and otheranimal species; and various bovine tissue protein extracts. Hybridomaswith distinct reaction patterns to bovine target TSPs were selected.Monoclonal antibodies (MAbs) showing positive reaction to antigens willinclude both IgM and IgG classes. Because IgM antibodies are generallymore difficult to purify and store, only IgG class of MAbs wereselected. This can be achieved by using IgG y-chain specific secondaryantibody as a probe in the ELISA screening procedures.

MAbs were obtained in supernatants from propagated cell cultures and inascites fluid from mice injected with hybridoma cells. The isotype ofthe selected MAb was determined with a mouse MAb isotyping kit (Sigma)according to the manufacturer's protocol. MAb IgGs were purified using aBio-Rad Protein A Cartridge with the Bio-Rad Econo LC system. Theconcentration of IgG in the final preparation was determined by UVabsorption at 280 nm. The purified MAbs were titrated against theantigen by indirect ELISA to confirm the immunoreactivity. The antigeniccomponents for each MAb were determined by Western blotting.

Characterization of Monoclonal Antibodies (MAbs) Indirect Enzyme-LinkedImmunoSorbent Assay (ELISA)

One hundred microliter (100 μL) of each sample protein extract andcontrols properly diluted in 0.06M carbonate buffer (pH 9.6) such thateach 100 μL contained 0.5 pg of soluble protein, was coated unto thewells of a 96-well polyvinyl microplate and incubated at 37° C. for 2hours. The plate was then washed 3 times with PBST [0.05% v/v Tween-20in 1 mM PBS, pH 7.2] and incubated with 200 μL/well blocking solution(1% BSA in PBS) at 37° C. for 2 h followed by another washing step. Theundiluted primary MAb supernatants (100 μL) or appropriately diluted inantibody buffer [1% w/v BSA in PBST] was added to each well andincubated at 37° C. for 2 hours. After washing 3 times with PBST,diluted (1:3,000 in antibody buffer) horseradish peroxidase-conjugatedgoat anti-mouse IgG-Fc specific solution was added. The plate wasincubated at 37° C. for 3 h and then washed 5 times before the additionof the substrate solution (22 mg of2,2′-azino-di-[3-ethyl-benothiazoline-6-sulfonic acid] and 15 μL of 30%hydrogen peroxide in 100 ml of 0.1 M phosphate-citrate buffer, pH 4.0).Color was developed at 37° C. for 20 min and the enzyme reaction wasstopped by adding 0.2 M citric acid solution (42 g citric acidmonohydrate dissolved in 1000 mL deionized water). Absorbance wasmeasured by a microplate reader (Bio-Rad, Model 450) at 415 nm. Thistechnique was used to test titers of the antisera, screen hybridomaclones, produce saturation curves for each MAb, test for specificity ofMAbs, and verify the cross reactivity of each MAb.

Table 1 summarizes the distinctive species-specific immunoreactivitiesof monoclonal antibodies to proteins extracted from heat-treated (cookedat 100° C. for 15 min or autoclaved at 121° C., 15 min, 1.2 bars) andnon-treated whole blood of different animal species (horse, pig, sheep,cattle, chicken, turkey and rabbit), and their cross-reactivity withseveral non-blood proteins (meat proteins, bovine serum albumin, gelatinand non-fat dry milk proteins) using indirect ELISA. All MAbs weredeveloped by immunizing animals with thermal-stable crude solubleprotein/peptide mixtures extracted from autoclaved bovine whole bloodwith 10 mM PBS as described in greater detail herein.

TABLE 1 Characterization of Monoclonal Antibodies Cross reactivitySpecies Specificity With MAb Autoclaved Cooked non-blood (Subclass)samples Samples Raw samples proteins 1B4 (IgG1) Equine ++++ Equine +++Equine +++ − Porcine ++++ Porcine +++ Porcine ++ Ovine ++++ Ovine ++++Ovine ++ Bovine ++++ Bovine ++++ Bovine ++ 1H9 (IgG1) Bovine ++++ Bovine++++ Bovine ++++ − Ovine +++ Ovine ++++ Ovine ++++ 2B11 (IgG1) Bovine++++ Bovine ++ Bovine ++ BSA ++++ Rabbit ++++ Rabbit ++ Rabbit + 3D6(IgG1) Bovine +++ Bovine +++ Bovine + − Equine ++++ Equine ++++ 6E1(IgG1) Bovine ++++ Bovine ++++ Bovine + − 6F10 (IgG1) Bovine ++++ Bovine++ Bovine + − Ovine +++ Ovine ++ 6G12 (IgG1) Bovine ++ Bovine ++Bovine + − Ovine ++++ Ovine ++++ 7F6 (IgG1) Bovine ++ Bovine ++ Bovine +− Ovine ++++ Ovine ++++ + = weak reaction, ++ = moderate reaction, +++ =strong reaction, ++++ = very strong reaction, − = negative reaction

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed to resolve the soluble TSPs of different bloodsample extracts. Western blot was then carried out to transfer proteinsfrom the gel to nitrocellulose membrane in order to determine themolecular weights of the immunogenic components which reacted withdeveloped MAbs. The general procedures of SDS-PAGE were performedaccording to the method of Laemmli (1970) with modifications. Briefly,soluble proteins from the samples were separated on 5% stacking gel (pH6.8) and a 12% polyacrylamide separating gel (pH 8.8). The gel waselectrophoresed at 200 V for 45 minutes using a Mini-Protein 3electrophoresis cell (Bio-Rad, 161-3301) with 1×Tris/Glycine/SDS buffer(100 mL 1×Tris/Glycine/SDS buffer in 900 mL DDI water, pH 8.3) connectedto a power supply (Bio-Rad Model 3000). Samples were diluted such thatthere was 3 μg of protein per each 15 μL of sample loaded into the well.

Table 2 summarizes the antigenic components in the blood proteinextracts probed by each monoclonal antibody produced using Western blot.

TABLE 2 Antigenic Components in the Blood Protein Probed by Certain MabsLaboratory prepared blood extracts & commercial plasma samples EstimatedMab Bba Sba Rba Hba Pba Bbm Bpm MW 1B4 ++++ ++++ − ++++ ++++ ++ ++ 25kDa 1H9 +++ ++ − − − ++ +++ 10 kDa 2B11 ++ − ++ − − − ++ 10 kDa 3D6 ++++− − ++++ + ++ ++++ 60 kDa 6E1 ++++ − − − − ++++ ++++ 40 kDa 6F10 ++ +++− − − − − 25 kDa 6G12 ++++ ++++ − − − ++ +++ 60 kDa 7F6 ++++ ++++ − − −++ +++ 60 kDa Bba = autoclaved bovine blood, Sba = autoclaved ovineblood, Rba = autoclaved rabbit blood, Hba = autoclaved equine blood, Pba= autoclaved porcine blood, Bbm = commercial bovine blood meal,commercial Bpm = bovine plasma meal, MW = molecular weight of antigenicprotein/peptide components. + = weak antigenic band, ++ = moderateantigenic band, +++ = strong antigenic band, ++++ = very strongantigenic band, − = negative antigenic band.

Western Blot

Western blots were performed according to Towbin and Others (1979) withmodifications. After separation of the proteins on 12% polyacrylamidegel by means of SDS-PAGE, protein bands were transferredelectrophoretically (1 h at 100V) from the gel to nitrocellulosemembranes using a MiniTrans-Blot unit (Bio-Rad) with 25 mM Tris, 192 mMglycine, and 20% (v/v) methanol buffer (pH 8.3). Upon completion of thetransfer, the membrane was washed with TBST (20 mM Tris, 500 mM NaCl,0.05% Tween-20, pH 7.5), blocked with 3% gelatin in PBS, and incubatedwith the undiluted MAb supernatant. The excess antibody was removed bywashing with TBST, and the membrane was incubated with goat anti-mouseIgG-alkaline phosphatase conjugate diluted 1:3,000 in antibody bufferfor 1 h at room temperature. After washing, the membrane was incubatedwith 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazoliumchloride (BCIP/NBT) in 0.1 M Tris buffer, pH 9.5. The color developmentwas observed within 10 to 20 min and reaction was stopped by washing themembrane with distilled water. The appearance of a dark purplish bandindicated the antibody binding site. The prestained broad range proteinstandards (Precision Plus Protein Kaleidoscope Standards, Bio-Rad,161-0375) were used as molecular weight markers in both SDS-PAGE andWestern blot.

Example 2 Development of a Sandwich ELISA for the Detection of BovineBlood in Animal Feedstuffs and in Ground Beef Characterization of MabsSpecificity and Cross-Reactivity

Species and tissues specificity of MAbs were determined by testing eachMAb supernatant with raw, cooked and autoclaved blood extracts fromdifferent animal species, meat extracts, and extracts from otherproteins using indirect ELISA procedures as described in Example I.

Epitope Comparison

Epitope comparison was performed to search two comparable MAbs forconstruction of a sandwich ELISA. The ELISA method developed by Friguetand others (1983) was adapted for comparison of the relative bindingsites of MAbs on the antigen. This test is based on an estimation of thelimited number of antigenic sites simultaneously available to a pair ofMAbs. The binding of the first MAb on the antigen inhibits the bindingof the second MAb if both bind to the same or relatively close epitopes.However, if a pair of MAbs recognizes different epitopes, then thereaction can be additive. The epitope comparisons between any two MAbcombinations were performed in two stages, namely generation ofsaturation curve to estimate the amount of each antibody required tosaturate the antigen coated on the wells followed by the additivity testusing indirect ELISA procedures.

To generate saturation curves, 200 μL of autoclaved bovine blood dilutedin carbonate buffer (pH 9.6) to correspond to a concentration of 0.5 μgprotein per 100 μL of coated sample, was coated into the wells of amicroplate and incubated for 2 hours at 37° C. After 3 washings, theplate was blocked with BSA and incubated at 37° C. for 2 hours. After 2further washings, 200 μL serial dilutions of MAb supernatant or purifiedIgG in antibody buffer (1% w/v BSA-PBST), were added to the plate andthe plate was incubated for 1 hour at 37° C. After washing 3 times, 100μL of horseradish peroxidase conjugated goat anti-mouse IgG (Fcspecific), diluted 1:3000 in antibody buffer was added to the plate andincubated for 1 hour at 37° C. The remaining color developmentprocedures were the same as described in the indirect ELISA. A plot withabsorbance on the y-axis and reciprocal of the antibody dilution on thex-axis was constructed to obtain the saturation curve. The dilution ofthe antibody required to saturate the coated antigen was obtained fromthe saturation curve for each antibody tested, as the point at which thecurve remains level or starts to drop.

To perform the additivity test for the two antibodies tested),autoclaved bovine blood extract (0.5 μg in 100 μL) diluted in carbonatebuffer (pH 9.6) was coated unto the wells of a microplate and incubatedfor 2 hours at 37° C. After 3 washings, the plate was, blocked with 200μL BSA solution and incubated at 37° C. for 2 hours. After 2 furtherwashings, 100 μL of the MAb #1 properly diluted in antibody buffer tosaturate the coated antigen (as determined from the saturation curve),was introduced into one well of the microplate. Another 100 μL of theMAb #2 sufficiently diluted to saturate the coated antigen (asdetermined from the saturation curve) was dispensed into the second wellof the microplate. One hundred microliter (100 μL) each of the first andsecond primary antibody at the same levels of dilution to saturate thecoated antigen were then introduced together into the third well of themicroplate. The plate was then incubated at 37° C. for 2 hours. Theremaining procedures of the ELISA were the same as described previously.The absorbance was read at 415 nm for the MAb #1 alone (A₁), the MAb #2alone (A₂) and the two MAbs together (A₁₊₂). The additivity index (A.I.)for the two MAbs was computed based on the formula below:

${A.I.} = {\left( {\frac{2A_{1 + 2}}{A_{1} + A_{2}} - 1} \right) \times 100}$

If the two MAbs bind randomly at the same epitope, A₁₊₂ should be equalto the mean value of A₁ and A₂ and A.I. will be equal to zero. If, onthe contrary, the two MAbs bind independently at distinct sites on theantigen molecule, A₁₊₂ should be the sum of A₁ and A₂ and A.I. will beequal to 100%. Thus for this experiment, for A.I. values below 50%, theMAbs were considered to inhibit each other's binding to the antigen,therefore, incapable of being used for the construction of a SandwichELISA. For A.I. values between 50% and 100%, the antibodies wereconsidered to bind to different epitopes on the antigen molecule andtherefore capable of being used to construct a Sandwich ELISA. In thissample experiment, MAb 3D6 and MAb 6G12 were selected to demonstrate thesandwich ELISA development.

MAb Purification and Biotinylation

Ascites fluids of selected MAbs were purified with Protein A column(Bio-Rad, 732-0091) in accordance with the manufacturer's instructionsusing the Econo System (Bio-Rad, 731-8101). The purified IgG (e.g. MAb3D6) was subjected to biotinylation by first dialyzing in 0.1MNaHCO_(3 [)16.802 g NaHCO₃ in 2 L deionized water with pH adjusted to8.3] for 24 hours at 4° C. The dialyzing buffer (0.1M NaHCO₃) waschanged four times within the period. The protein concentration of thedialyzed antibody was determined by determining the absorbance at 280 nm(the UV assay) using the SmartSpec 3000 spectrophotometer (Bio-Rad).Ten-milligram (10 mg) NHS-CA-Biotin (Biotinamidocaproic acid3-sulfo-N-hydroxysuccinimide ester) was dissolved in 1 mL DMF(N,N-Dimethyl Formamide) and added to the dialyzed antibody such that toeach mg of dialyzed antibody, 100 μg of NHS-CA-Biotin in DMF was added.The mixture was incubated at room temperature for 1 hour and dialyzedovernight in 1 mM PBS at 4° C. The protein concentration of thebiotinylated antibody was determined by UV assay prior to storage at−20° C.

Sample Preparations Preparation of Flesh (Meat) and Non-Flesh ProteinSamples

COOKED AND AUTOCLAVED MEAT EXTRACTS—Fat and connective tissues weretrimmed off meat samples (beef, pork, lamb, rabbit, horse, chicken, andturkey). The lean meat sample was then cut up and ground twice using ameat grinder (Waring consumer Products, East Windsor, N.J.) to ensurethoroughness and homogeneity. Ten-gram (10 g) of minced meat sample fromeach species was weighed into beakers. The beakers were covered withaluminum foil, sealed with adhesive tape and autoclaved for 15 minutesat 121° C. and a pressure of 1.2 bars. Another portion (10 g) of mincedmeat sample from each species was weighed into a beaker. The beaker wascovered with aluminum foil and the heated in a boiling water bath for 15minutes. The autoclaved and cooked samples were then mashed into fineparticles using a glass rod. Ten-milliliter (10 mL) of extraction buffer(10 mM PBS) was then added to the mashed autoclaved and cooked samplesto extract the soluble proteins. The mixture was then homogenized for 2minutes at 11000 RPM. The homogenized sample was then kept at roomtemperature for 2 hours followed by another 2 hours at 4° C. Themixtures were transferred in to centrifuge tubes and centrifuged at3220×g for 60 minutes at 4° C. The supernatant was then filtered throughWhatman No. 1 filter paper. The clear filtrates were used as theautoclaved and cooked meat sample extracts for the sandwich ELISA.

RAW BEEF EXTRACT—Ten-gram (10 g) of minced raw beef was weighed into asampling bag. Ten-milliliter (10 mL) of extraction buffer (10 mM PBS)was added to extract the soluble proteins. The mixture in the samplingbag was blended in a stomacher (Model Number STO 400, Tekmar Company,Cincinnati, Ohio) for 60 seconds and allowed to stand at roomtemperature for 2 hours followed by another 2 hours at 4° C. The mixturewas transferred in to a centrifuge tube and centrifuged and filtered asdescribed above. The clear filtrates were used as raw meat extract forthe sandwich ELISA.

NON-FLESH PROTEINS—The non-flesh proteins used were gelatin, soy proteinconcentrate, egg albumin, nonfat dry milk, Dairy Blend and bovine serumalbumin. To 2 g each of the non-flesh proteins in a beaker was added 10mL of extraction buffer (10 mM PBS) to extract the soluble proteins. Themixtures in beakers were covered with parafilms and kept at 2 hours atroom temperature followed by another 2 hours at 4° C. The mixtures werethen transferred into centrifuge tubes and centrifuged at 3220×g for 60minutes at 4° C. The supernatant was filtered through Whatman No. 1filter paper and the clear filtrates were used in the sandwich ELISA.

Preparation of Commercial Feedstuffs

To 2 g each of bovine meat bone meal, sheep meat bone meal, porcine meatbone meal, feather meal, spray-dried bovine plasma, whole bovine bloodpowder, and spray-dried porcine plasma was added 10 mL of extractionbuffer (10 mM PBS) to extract the soluble proteins. The mixtures wereextracted, centrifuged and filtered as described above.

Preparation of Laboratory Adulterated Feed and Ground Beef Samples

Artificially adulterated samples used in this experiment were 1)autoclaved bovine blood in autoclaved porcine blood; 2) spray-driedbovine plasma and whole bovine blood powder in spray-dried porcineplasma; and 3) bovine blood in raw and cooked ground beef.

Autoclaved bovine blood in autoclaved porcine blood adulterated sampleswere prepared by mixing autoclaved porcine blood extract with autoclavedbovine blood extract to give 0 to 10% (v/v) adulterated samples with 0%been autoclaved porcine blood with no added autoclaved bovine blood.Likewise, the spray-dried bovine plasma in spray-dried porcine plasmaadulterated samples were prepared by mixing spray-dried porcine plasmaextract with spray-dried bovine plasma extract to give a 0 to 10% (v/v)level of adulteration. Whole bovine blood powder in spray-dried porcineplasma adulterated samples were prepared by mixing spray-dried porcineplasma extract with whole bovine blood powder extract to give a 0 to 10%(v/v) level of adulteration.

Preparation of Laboratory Adulterated Ground Beef Samples

Cooked and raw ground beef adulterated with bovine blood, were preparedin two ways. In the first case, prepared cooked ground beef extract wasmixed with cooked bovine blood extract to give a 0 to 10% v/v bovineblood in cooked ground beef, with 0% being cooked ground beef extractwithout added cooked bovine blood. Bovine blood in raw ground beef wasprepared similarly by mixing raw ground beef extracts with raw bovineblood to give a 0 to 10% v/v spiked bovine blood in raw ground beefextracts, with 0% being raw ground beef without added raw bovine blood.

Alternatively, samples were prepared by mixing bovine whole blood withfresh ground beef at 0% to 50% (v/w) levels. Ten grams of ground beefwas weighed into beakers, a volume of 100 μL, 300 μL, 500 μL, 1 mL, 2mL, 3 mL, 4 mL and 5 mL of bovine blood was added to each beaker to give1, 3, 5, 10, 20, 30, 40 and 50% v/w spiked bovine blood in ground beefsamples, respectively. The blood and beef mixture was thoroughly mixed,covered with aluminum foil, and heated in boiling water bath for 15minutes. After cooling, 10 ml of extraction buffer (10 mM PBS) was addedand the mixture was homogenized for 2 minutes at 11000 RPM. The mixtureswere covered with paraffin films and allowed to stand for 2 hours atroom temperature followed by another 2 hours at 4° C. The mixtures werecentrifuged at 3220×g for 60 minutes at 4° C. The supernatant wasfiltered through Whatman No. 1 filter paper and the clear filtrate wasused as cooked sample extract for that sandwich ELISA.

Adulterated blood in raw beef extracts were mixed the blood and groundbeef in a similar fashion as the cooked samples. The mixtures weretransferred into sampling bags and to this was added 10 mL of extractionbuffer. The mixtures were then blended in a stomacher for 60 seconds andallowed to stand at room temperature for 2 hours followed by another 2hours at 4° C. The mixtures were transferred into centrifuge tubes andcentrifuged and filtered and the clear filtrate was used as the rawblood adulterated beef sample extracts for analysis.

Sandwich ELISA

The experimental conditions including incubation time, titers ofantibodies, and sample dilution of the sandwich ELISA were optimized toachieve the highest sensitivity and detectability. One hundredmicroliter (100 μL of the capture antibody (MAb 6G12 purified IgG)diluted 1:1000 in antibody buffer (1% BSA-PBST), which corresponds to0.18 μg protein per 100 μL was coated on the wells of a microplate andincubated at 37° C. for 2 hours. The plate was washed three times withPBST and then incubated for 1 hour at 37° C. with 200 μL of blockingbuffer (1% BSA-PBS). After washing the plate twice with PBST, 100 μL ofcontrols and samples appropriately diluted in antibody buffer was addedto the plate and the plate was incubated for 2 hours at 37° C. The platewas washed thrice and incubated with 100 μL of the detection antibody(biotin-conjugated 3D6) diluted 1:1000 (which corresponds to 0.175 μgprotein per 100 μL) in antibody buffer for 2 hours at 37° C. Afterfurther washing three times, the plate was incubated for 1 hour at 37°C. with 100 μL of the enzyme, streptavidin peroxidase polymer. Afteranother washing step for five times, 100 μL of the enzyme substrate(ABTS with added H₂O₂) was added to the wells and the color wasdeveloped for 20 minutes at 37° C. At the end of the 20 minutes, 100 μLof stop solution (0.2M citric acid solution) was added to stop thereaction. The absorbance was then read at 415 nm.

Validation of the Sandwich ELISA

The performance of the optimized sandwich ELISA using MAbs 3D6 as thecapturing antibody and MAb 6G12 as the detecting antibody was evaluatedand validated in term of the criteria including the limit of detection,intra-assay and inter-assay variability (reproducibility), sensitivity,specificity and overall accuracy.

The limit of detection is the smallest quantity of the analyte that canbe reliably distinguished from the background noise in the test (Dixon1998). This was determined using adulterated commercially producedfeedstuffs as well as laboratory prepared blood samples. See, FIGS. 8-10and 13-18.

Reproducibility is the variability between replicate determinations inthe same assay (intra-assay variability) and in different assays(inter-assay variability) and this is represented as the coefficient ofvariation (CV). This was done using bovine blood containing samples(spray-dried bovine plasma, whole bovine blood powder, autoclaved,cooked and raw bovine blood) at levels above the detection limit.

Sensitivity is the ability of the test to detect positive samples aspositive and was computed as A/B×100% where B was the number of positivesamples tested and A the number of positive samples that the test wasable to correctly detect as positive. Specificity is the ability of thetest to detect negative samples as negative and was computed as C/D×100%where D was the number of negative samples tested and C the number ofnegative samples that the test was able to correctly detect as negative.Overall accuracy is the combined ability of the test to correctly detectpositive and negative samples (overall accuracy=specificity andsensitivity) (Dixon 1998). This was computed as E/F×100% where F was thetotal number of positive and negative samples tested and E the number ofpositive and negative samples correctly detected by the assay. Allbovine blood containing samples (spray-dried bovine plasma; whole bovineblood powder; autoclaved, cooked and raw bovine blood) at concentrationlevels above the detection limit were used to compute the overallaccuracy of the sandwich ELISA.

Statistical Analysis

All experiments were performed in triplicate and results analyzed usingMicrosoft Excel 2000. One-way ANOVA using SPSS software (11.0 forWindows) (SPSS Inc., Chicago, Ill.) was used to compare means fordifference among three or more treatment groups. Post-hoc analysis wasperformed using Turkey HSD. Paired T-test was used to compare means ofbackground noise and detection limit. Significance was accepted atp≦0.05.

1. An immunoassay method for determining presence of bovine blood in asample, the method comprising the steps of: combining the sample with afirst monoclonal antibody having an affinity for a thermostable bovineblood antigen, wherein the monoclonal antibody is selected from thegroup consisting of 1B4, 1H9, 2B11, 3D6, 6E1, 6F10, 6G12 and 7F6produced by hybridoma cell lines deposited as ATCC Nos. PTA-9870,PTA-9869, PTA-9868, PTA-9867, PTA-9982, PTA-9983, PTA-9985 and PTA-9984,respectively; and detecting the formation of a complex between themonoclonal antibody and the bovine blood antigen, thereby detecting thepresence of bovine blood in the sample by detecting a label that isattached to (a) the monoclonal antibody; (b) a second monoclonalantibody that is also selected from the group consisting of 1B4, 1H9,2B11, 3D6, 6E1, 6F10, 6G12 and 7F6; or to (c) a secondary antibodyhaving affinity for the first monoclonal antibody.
 2. The method ofclaim 1, wherein the method is carried out as a sandwich assay in whichthe first monoclonal antibody is bound to a carrier, the bound carrieris combined with the sample, and the label is bound to the secondmonoclonal antibody selected from the group consisting of 1B4, 1H9,2B11, 3D6, 6E1, 6F10, 6G12 and 7F6.
 3. The method of claim 2, whereinthe first and second monoclonal antibodies are different monoclonalantibodies selected from the group consisting of 1B4, 1H9, 2B11, 3D6,6E1, 6F10, 6G12 and 7F6 produced by hybridoma cell lines deposited asATCC Nos. PTA-9870, PTA-9869, PTA-9868, PTA-9867, PTA-9982, PTA-9983,PTA-9985 and PTA-9984, respectively.
 4. The method of claim 3, whereinthe first monoclonal antibody is 6G12 and the second labeled monoclonalantibody is 3D6.
 5. An immunoassay method for determining presence ofbovine blood in a food sample or an animal feed sample, the methodcomprising the steps of: coating a carrier surface with a samplepotentially containing bovine blood antigen: contacting the coatedsurface with a first monoclonal antibody having affinity for athermostable bovine blood antigen, wherein the monoclonal antibody isselected from the group consisting of 1B4, 1H9, 2B11, 3D6, 6E1, 6F10,6G12 and 7F6 produced by hybridoma cell lines deposited as ATCC Nos.PTA-9870, PTA-9869, PTA-9868, PTA-9867, PTA-9982, PTA-9983, PTA-9985 andPTA-9984, respectively; and determining whether the first monoclonalantibody bound to the coated surface.
 6. The method of claim 5 whereinthe first monoclonal antibody is labeled with a reporter molecule. 7.The method of claim 5, wherein said determination is carried out using asecondary antibody labeled with a reporter molecule, the secondaryantibody having an affinity for the first monoclonal antibody.
 8. Themethod of claim 5, wherein the first monoclonal antibody is 6E1.